Progestin-primed Ovarian Stimulation With Clomiphene Citrate Compared With the Standard Progestin-primed Ovarian Stimulation in Various Aged Women With Diminished Ovarian Reserve: a Retrospective Study

Background: Clomiphene citrate (CC) supplementation in the progestin-primed ovarian stimulation (PPOS) protocol effectively alleviated profound pituitary suppression from progestin administration and reduced the gonadotropin (Gn) dose in the population of women with normal-ovarian-reserve or polycystic ovarian syndrome. Limited data are available about the role of CC in PPOS for women with diminished ovarian reserve (DOR). This study aimed to compare the eciency of the PPOS protocol with CC supplementation and the standard PPOS protocol for various aged women with DOR. Methods: A total of 364 patients with DOR were recruited for this retrospective cohort study. They were divided into subgroups based on female age, ≤ 35 years and (cid:0)35 years. The clinical characteristics and outcome were compared between the groups in subgroups. Results: In all patients with DOR, the PPOS protocol with CC supplementation was associated with a lower percentage of women with profound pituitary suppression (0.0% vs 18.6%, P (cid:0)0.001 and 1.3% vs 11.0%, P (cid:0)0.002) and a higher mean luteinizing hormone (LH) level during controlled ovarian stimulation (COS) than the standard PPOS protocol (P (cid:0)0.05(cid:0). In young women with DOR, more number of high-quality cleavage-stage embryos (1.96 vs. 1.38, P (cid:0)0.018) was achieved in the PPOS protocol with CC supplementation. In elderly women with DOR, PPOS protocol with CC supplementation led to an increase in the incidence of LH levels above 10 IU/L on the trigger day (12.7% vs. 4.9%, P (cid:0)0.028) and decrease in the rate of oocyte maturation (84.7% vs. 89.9%, P (cid:0)0.034) compared to the standard PPOS protocol. No signicant differences were observed in the Gn duration, total dosage of Gn, and pregnancy outcomes between the groups. Conclusions: CC is an effective adjuvant to alleviate pituitary suppression in the PPOS protocol. For young women with DOR, CC supplementation has a positive impact on the number of high-quality embryos in PPOS protocol. However, elderly patients with DOR would be at risk of developing premature LH surge and poor oocyte maturation rate after the PPOS protocol with CC supplementation was applied.


Introduction
In assisted reproduction technologies (ART), the number of retrieved oocytes largely depends on a woman's ovarian reserve. Diminished ovarian reserve (DOR), which can be age dependent or independent, are manifested mainly as decreased anti-Müllerian hormone (AMH) and low antral follicle count (AFC) [1]. Patients with DOR may be at risk of poor ovarian response, especially in women aged 35 to 40 [2]. A low or poor ovarian response often results in cycle cancellation, poor outcomes, and consequent stress and disappointment to the couple. Such patients could bene t from counseling regarding protocol selection to improve the clinical outcomes [3]. Therefore, clinicians and researchers worldwide are seeking a proper controlled ovarian stimulation (COS) protocol for patients with DOR.
Progestin-primed ovarian stimulation (PPOS) has been approved its effective in preventing an endogenous luteinizing hormone (LH) surge during COS [4][5][6][7]. But the continuous supply of progestin during PPOS can lead to profound pituitary suppression [4][5][6]. It was reported that profound pituitary suppression was positively correlated with the gonadotropin (Gn) duration [5]. Clomiphene citrate (CC) was used to block negative feedback trigged by estrogen, thereby resulting in elevated follicle-stimulating hormone and LH release from the hypothalamus [8,9].
PPOS supplemented with CC signi cantly reduced the occurrence of profound pituitary suppression in women with normal ovarian reserve or polycystic ovarian syndrome and the combination of CC and PPOS is likely to reduce the Gn duration compared to those without CC [5,6].
Currently, limited data are available about the role of CC in PPOS for women with DOR. In addition, age is considered a marker for oocyte quality, which is correlated with embryo euploid rates in ART [10]. Therefore, the present study was designed to compare the e ciency of the PPOS protocol with CC supplementation and the standard PPOS protocol for various aged women with DOR.

Study design and participants
We conducted a retrospective cohort study at the reproductive medicine center of the First A liated Hospital of Wenzhou Medical University. From June 2018 to February 2020, women with two or more of the following items were diagnosed with DOR: (a) FSH ≥ 10IU; (b) FSH/LH ≥ 2; (c) AFC ≤ 8; and (d) AMH ≤ 1.1. Both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles were included. Women who were diagnosed with uterine cavity abnormalities, untreated hydrosalpinx, and immunologic disease were excluded. Every woman contributed to the study with a single stimulation cycle only. In women with multiple treatment cycles, the chronologically rst cycle was chosen. For analysis, couples were grouped as follows: patients using PPOS protocol with CC supplementation were grouped into the "PPOS with CC group", while patients using standard PPOS protocol were grouped into the "standard PPOS group". Subpopulations of patients were characterized by different ranges of age, ≤35 years and 35 years. All data were retrospectively collected from computer databases and stored in a deidenti ed database.
Permission for this study was given from the ethics committee of the First A liated Hospital of Wenzhou Medical University (no. 2020-02).

Stimulation protocols and pituitary suppression
In the PPOS with CC group, HMG (Shanghai Livzon Pharmaceutical Company, Zhuhai, China) 150-225 IU, CC (Codal Synto Limited, Limassol, France) 100 mg and MPA (Shanghai Xinyi Pharmaceutical Co., China) 10 mg daily were administered from menstrual cycle day 2-3 (MC2-3) or after an episode of withdrawal bleeding. In standard PPOS group, HMG 150-225 IU and MPA 10 mg daily were initiated from menstrual cycle day 2-3 (MC2-3) or after an episode of withdrawal bleeding. The initial dose of Gn (150-225 IU) was based on patient's age, basic FSH, AFC, and previous promotion plan. Follicle monitor and hormone assay (FSH, LH, E 2 and P) were performed 5 days later. The dose of Gn was adjusted according to follicle development and MPA dose was consistent up to the trigger day.
When the dominant follicle diameter reached 17 mm and the majority of growing follicles, if there were any, reached 14mm or more, recombinant human chorionic gonadotropin (Ovitrelle, Serono, Bari, Italy) were administered to trigger ovulation. Oocytes were retrieved 36 to 38 hours later. All follicles larger than 10 mm in diameter were aspirated.
Fertilization was carried out in vitro after oocyte retrieval depending on the semen parameters and previous fertilization situation. Embryos were examined for the number or regularity of blastomeres and the degree of fragmentation. One or two high-quality cleavage-stage embryos (7-9 cells and 20% fragmentation) were frozen via vitri cation on the 3rd day after oocyte retrieval, and remaining embryos were placed in extended culture [11,12].
Subsequently, blastocysts with good morphological grades were frozen on day 5 or 6 of culture.

Hormone measurement
Serum FSH, LH, E 2 , and P were measured on menstrual cycle day 2-3, day 7-10, and the trigger day. Hormone levels were determined with immuno uorescence assay (Roche Diagnostics, Mannheim, Germany). The lower limits of sensitivity were as follow: FSH 0.06 IU/L, LH 0.09 IU/L, E 2 10 pg/ml and P 0.1 ng/ml. A serum LH concentration 1.0 IU/L on the trigger day was set as the cut-off for profound pituitary suppression [13].

Endometrium preparation and Frozen embryo transfer
All patients in the present study had received rozen-thawed embryo transfer (FET). Hormone replacement treatment was used for endometrial preparation. Brie y, the timing of embryo transfer was scheduled on the 3rd or the 5th day after progestin administration depending on the embryo stage. Embryo transfer was all conducted via the guidance of abdominal ultrasound in our center. Each patient received no more than two embryos at one time. Patients received luteal support after embryo transfer in the form of intravaginal progestin 200mg two times daily. Once pregnancy was achieved, the luteal support was continued until 10 weeks of gestation.

Outcome measures
We analyzed the pregnancy outcomes of the chronologically rst cycle after oocyte retrieval that transferred at least one high-quality cleavage-stage embryos or good morphology blastocysts (blastocysts better than grade 322) [11].
Clinical pregnancy was de ned as the presence of fetal cardiac activity con rmed by transvaginal ultrasound.
Ongoing pregnancy rate was de ned as the proportion of patients with ongoing pregnancy after the gestation age of 12 weeks. The implantation rate was calculated as the number of gestational sacs visualized on transvaginal ultrasound divided by the number of transferred embryos.

Statistical analysis
The data were evaluated by Student's t-test for continuous variables of normal distribution, the Mann-Whitney U-test for continuous variables of non-normal distribution, the χ 2 -test or Fisher's exact for categorical variables, as appropriate. All tests were two-sided, and P 0.05 was considered statistically signi cant. SPSS statistical software (version 25; SPSS Inc., USA) was used for data analysis. The dynamic changes in hormones during COS were presented with a broken line graph, created by Excel.

Patient characteristics and cycle characteristics
In total, 364 women were enrolled. Table 1 shows the comparison of basic patient characteristics and the cycle characteristics between ovarian stimulation groups strati ed by age. No statistically signi cant differences were found between the groups with respect to age, duration of infertility, proportion of secondary infertility, body mass index (BMI), AMH, AFC, baseline hormones, Gn duration and total dosage of Gn (P > 0.05).  Hormone pro le during treatment The serum concentrations of FSH, LH, E 2 and P in the two groups strati ed by age are presented in Figure 1. In both groups, the FSH levels increased after Gn administration, and no difference was found between the groups (P 0.05).
The serum E 2 levels and P levels increased gradually in both groups after Gn administration. The LH levels of the two groups showed different trends. The LH levels in the PPOS with CC group increased rst and then remained steady at a range of 5.52-6.61 IU/L; in contrast, the LH levels in the standard PPOS group remained at a certain level initially and then slightly decreased. There was no signi cant difference in the value of basal LH between the groups (P 0.05), whereas the LH levels on day 7-10 and day of trigger in the PPOS with CC group were signi cantly higher than that in all subgroups of the standard PPOS group (P 0.05).
As shown in Table 1, the percentage of women with profound pituitary suppression was signi cantly lower in the PPOS with CC group than that in standard PPOS group (0.0% vs 18.6%, P 0.001 and 1.3% vs 11.0%, P =0.002). In addition, compared with the standard PPOS group, the percentage of patients with LH levels above 10 IU/L on the trigger day was higher in the PPOS with CC group, especially for patients aged above 35 years (12.7% vs. 4.9%, P 0.028). All patients with LH levels above 10 IU/L on the trigger day con rmed the sonolucent follicles are still there by transvaginal ultrasound just before the planned oocyte retrieval and underwent oocyte retrieval, among which only two patients had no oocytes retrieved and only one patient had no mature oocytes being retrieved.

Pregnancy outcomes
In this study, 29 women did not complete the FET cycles for no viable embryos and 62 women for personal reasons before the end of the study. In total, 273 women completed FET cycles during the 18 months of follow-up, among which 4 women transferred with embryos collected from twice oocyte retrieval and 29 women transferred with no high-quality cleavage-stage embryos or good morphology blastocysts. The discontinuation rate of the cycle, which achieved at least one high-quality cleavage-stage embryos, was comparable between the groups ( All newborns were examined with no congenital malformation except that oesophageal atresia was found in one baby of the PPOS with CC group and cleft lip and palate in one baby of the standard PPOS group. Furthermore, we assess the clinical outcomes of patients with LH levels above 1 IU/L, but below 10 IU/L on the trigger day (Table 3). We found similar trend of the outcomes in these patients as in the whole population. Table 3 Clinical outcomes patients with LH levels above 1 IU/L, but below 10 IU/L on the trigger day between groups strati ed by age.

Discussion
This retrospective cohort trial showed that the percentage of women with profound pituitary suppression was signi cantly lower in the PPOS with CC group than that in standard PPOS group in all subgroups. For young women with DOR, PPOS protocol with CC supplementation led to an increase in the number of high-quality cleavage-stage embryos compared to the standard PPOS protocol. For elderly patients with DOR, the e ciency of the two protocols in embryological outcomes was similar with each other, but signi cantly higher incidence of LH levels above 10 IU/L on the trigger day and signi cantly lower rate of oocyte maturation were found in the PPOS with CC group.
According to progesterone's antipositive feedback mechanism, concurrent administration of progestin with estrogen inhibited estrogen's positive feedback and therefore abolished the premature LH surge [14,15]. Kuang et al [4] indicated that the application of progestin from the beginning of ovarian stimulation may lead to profound pituitary suppression and women in the standard PPOS protocol exhibited lower LH levels and had higher rates of profound pituitary suppression compared with the PPOS protocol with CC supplementation. According to our results, the proportion of women with profound pituitary suppression was also signi cantly higher in the standard PPOS protocol than in the PPOS protocol with CC supplementation in all subgroups (P 0.01  [17] in patients with very low LH concentrations ( 0.5 IU/L). Low LH Level on the day of trigger ( 1.60 mIU/ml) was reported to be associated with reduced ongoing pregnancy and live birth rates and increased early miscarriage rates [19]. CC interacts with the hypothalamic estrogen receptors and increases endogenous FSH and LH secretion by blocking estrogen's negative feedback mechanism [8, 20,21]. In this study, the LH levels during the COS process were higher in the PPOS with CC group in all subgroups (P 0.01). Therefore, CC supplementation alleviated profound LH suppression.
Our data showed that the LH levels on day 7-10 and day of trigger in the PPOS with CC group were signi cantly higher than those in all subgroups of the standard PPOS group (P 0.05). The key role of LH in synthesizing and secreting androgens, which are required for further production of E 2 and P, is widely acknowledged [22]. In the present study, E 2 levels and P levels at the time of ovulation were signi cantly higher in the PPOS with CC group than in the standard PPOS group (P 0.01), indicating that a high level of LH in the PPOS with CC group may further promote E 2 and P synthesis. According to the concept of a therapeutic window for LH, proposed by Hillier, there is a threshold requirement for LH for an optimal cycle outcome [23]. In the present study, the number of high-quality cleavage-stage embryos was higher among younger patients treated with the PPOS protocol with CC supplementation. Whereas, HMG was used in both treatments and we noted that Gn duration, total dosage of Gn, and the FSH level during COS between the groups were similar. It was not likely that the improved embryological outcomes could be attributed to the Gn use in the PPOS with CC group. These ndings further support the notion that LH plays a critical role in normal follicular development [24]. However, for elderly patients with DOR, though the number of 14mm follicles on trigger day was comparable between the groups (P 0.05), PPOS protocol with CC supplementation led to a decrease in the rate of oocyte maturation compared to the standard PPOS protocol. In addition, the embryological outcomes between the groups were similar in elderly patients with DOR. We found similar trend of the outcomes in patients with LH levels above 1 IU/L, but below 10 IU/L on the trigger day as in the whole population. Furthermore, the pregnancy outcomes were comparable between the groups, demonstrating a similar development potential of the embryos despite the different LH levels. Thus, our study indicated that elevated serum LH during the COS process had no impact on embryological outcomes, but might be associated with decreases in the oocyte maturation rate for elderly patients with DOR. The possible explanation is that women with DOR of different ages may require different range of therapeutic window for LH [24]. On the basis of these results, we suggest that the early stage of embryo development may differ slightly between oocytes retrieved after the PPOS protocol with CC supplementation or standard PPOS protocol was applied. Additional fundamental researches should be performed to determine the alterations in the follicular microenvironment, which may help determine the optimal range of the LH level, elucidate the mechanism by which LH affects oocyte quality and provide evidence for the use of PPOS.
Our data showed that the incidence of profound pituitary suppression was lower but the incidence of LH levels above 10 IU/L on the trigger day was higher in the PPOS with CC group, especially for patients aged above 35 years (P 0.05). Guo et al reported that higher percentage of premature LH surge (10.2%) was found in patients aged more than 35 years after the PPOS protocols using utrogestan applied [25]. Three other independent previous studies have found a similar correlation between low ovarian reserve or older age group patients and premature LH elevation [26][27][28]. It suggested that, though CC supplementation alleviated profound LH suppression, elderly patients with DOR would be at risk of developing premature LH surge and poor oocyte maturation rate.
Our study had several strengths as well as a few limitations. This trial lls a gap in the literature because there are relatively few published studies verifying the feasibility of CC co-administration in the PPOS protocol in various aged women with DOR. These results from a CC combination regimen may provide a new insight for develop an individualized treatment regimen for women with DOR to improve clinical outcomes. Because we used the "freezeall" strategy, there was a waiting period between oocyte retrieval and embryo transfer. Though there are 116 extra high-quality cleavage-stage embryos or good morphology blastocysts dose not get transferred, because the cycle discontinuation rate was similar between the groups, any possible bias should be minimal.

Conclusions
This retrospective cohort trial demonstrated that CC supplementation could mitigate the profound LH suppression caused by progestin administration. For young women with DOR, PPOS protocol with CC supplementation led to an increase in the number of high-quality cleavage-stage embryos compared to the standard PPOS protocol. However, elderly patients with DOR would be at risk of developing premature LH surge and poor oocyte maturation rate after the PPOS protocol with CC supplementation was applied.